The continued development of new tools and techniques that improve sensitivity, selectivity, speed and throughput in biological analysis, while reducing the cost per assay, will be critical in clinical diagnosis, medical research, and other disciplines in the life sciences. Micromachining methods have the potential to enable the construction of low-cost, yet sophisticated microfluidic systems for the analysis of complex protein mixtures. Herein, we propose to develop a phase-changing sacrificial layer (PCSL) microfabrication approach and polymer surface modification methods to create inexpensive microfluidic devices that can improve the detection and quantification of alpha-fetoprotein (AFP), a cancer and fetal wellness marker, in blood and urine. The objectives of this proposal are four-fold. First, we will generalize the PCSL solvent bonding fabrication technique and atom-transfer radical polymerization surface modification methods to a range of polymer microfluidic systems for protein analysis. Second, we plan to evaluate the PCSL solvent bonding approach for making complex, multilayer microfluidic arrays that facilitate the integration of sample processing steps. Third, we will develop these fabrication techniques to enable the incorporation of immunoaffinity purification systems and protein sample preconcentrators for use in enhancing detection in microchip capillary electrophoresis. Fourth, we will utilize the tools from the first three objectives to make microdevices that extract and preconcentrate lower-abundance proteins such as AFP from biological mixtures;we will then evaluate these microsystems for the determination of AFP levels in blood and urine. Importantly, while the studies in this proposal are directed toward the development of miniaturized assays for AFP, these same fabrication and analysis procedures can be generalized in a straightforward manner to allow the rapid quantification of other lower-abundance cancer and disease biomarkers. Thus, PCSL techniques and polymer surface derivatization methods should provide a broadly applicable microchip platform for the electrophoretic analysis and quantification of proteins or other biomolecules in humans.